Takara RetroNectin EIA Kit \96 Assays

This kit is a sandwich ELISA using as the capture antibody a plate-coated RetroNectin-specific monoclonal antibody that does not cross-react with human fibronectin. It is not subject to inhibition even in the presence of human blood components, allowing easy monitoring of the level of RetroNectin. This kit allows highly sensitive quantitative determination of release from RetroNectin-coated plates and residual RetroNectin levels in both cryopreservation solutions of cells cultured in a RetroNectin-added medium and cell washings.

Required Products

License Statement

RetroNectin reagent is a 63-kD fragment of recombinant human fibronectin fragment (also referred to as rFN-CH-296) that enhances the efficiency of lentiviral- and retroviral-mediated gene transduction. This is particularly important for hematopoietic cells and other hard-to-transfect cell types. When coated on the surface of containers such as culture dishes, petri dishes, flasks or bags, RetroNectin significantly enhances virus-mediated gene transduction into mammalian cells. This enhanced transduction is thought to result from the colocalization of virus particles and target cells on the molecules of RetroNectin. Viral particles bind RetroNectin via interaction with heparin-binding domain II, and target cells bind mainly through the interaction of the cell surface integrin receptor VLA-4 with the fibronectin CS1 site.

Schematic protocol for infecting target cells using RetroNectin reagent.In the RBV (RetroNectin-bound virus) method, the retrovirus is first bound to the RetroNectin-coated plate, and cells are added after removing the retrovirus supernatant. In this protocol, removal of the supernatant reduces inhibitory molecules that otherwise can reduce the transduction efficiency.

Comparison of T cell expansion efficiencies for various commonly used protocols.Cells co-stimulated using RetroNectin and α-CD3 demonstrated higher-fold expansion as compared to other protocols(Panel A). In addition, the presence of RetroNectin promoted the production of more naïve-like T cells (CCR7+/CD45RA+ phenotype) that have been shown to have better in vivo anti-tumor activity(Panel B).

NOTE:This expansion method requires a license for commercial applications.

Comparison of retroviral transduction of a K562 cell line using transduction enhancers from different companies.A K562 cell line was transduced with a retroviral vector in the presence of various transduction enhancers. RetroNectin yielded a far higher transduction efficiency relative to each of the other transduction enhancers, including Polybrene, protamine sulfate, and human fibronectin from three other companies.

Efficient transduction of human PBMCs using RetroNectin, as compared to Polybrene.Human PBMCs were transduced with varying dilutions of lentivirus encodingZsGreen1in the presence of RetroNectin or Polybrene, and the percentages of transduced CD8+ cells were measured by flow cytometry. Transduction efficiencies with Retronectin were significantly higher than those obtained using Polybrene, especially at low MOI (90X dilution).

T-cell expansion using RetroNectin reagent.Peripheral blood T cells were stimulated and expanded in vitro using anti-CD3 (clone OKT3) and IL-2 alone or in combination with RetroNectin reagent (RN), anti-CD28, or anti-4-1BB. Stimulation with RetroNectin reagent yielded higher proportions of cytotoxic CD8+ T cells.

RetroNectin reagent is a recombinant human fibronectin fragment that contains three functional domains: the cell-binding domain (C-domain), the heparin-binding domain (H-domain), and the CS-1 sequence.RetroNectin Reagent enhances lentiviral- and retroviral-mediated gene transduction by aiding the colocalization of target cells and viral particles. Specifically, virus particles bind RetroNectin Reagent via interaction with the H-domain, and target cells bind mainly through the interaction of cell surface integrin receptors VLA-5 and/or VLA-4 with the fibronectin C-domain and CS-1 sites, respectively. By facilitating close physical proximity, RetroNectin Reagent can enhance viral-mediated gene transfer to target cells expressing integrin receptors VLA-4 and/or VLA-5.